The present invention relates to a process for microbiologically producing reduced nicotinamide adenine dinucleotide (hereafter simply referred to as NADH) oxidase which participates in the enzyme reaction which oxidizes NADH to form hydrogen peroxide or water. More specifically, the present invention relates to a process for efficiently producing NADH oxidase having excellent temperature stability by genetic engineering and a plasmid used for the process as well as a transformed microorganism.
Nicotinamide adenine dinucleotide (hereafter simply referred to as NAD.sup.+) is a coenzyme of various dehydrogenases and receives hydride ions from the corresponding substrate by the action of dehydrogenase, that is, oxidizes the corresponding substrate and NAD.sup.+ itself is reduced to NADH. The formed NADH undergoes oxidation by the action of NADH oxidase, using water or molecular oxygen as a hydrogen receptor, whereby NAD.sup.+ is produced.
By allowing the dehydrogenase and NADH oxidase to co-exist, the regeneration system of NAD.sup.+ is formed and dehydrogenase reaction of industrially useful optically active compounds or reaction intermediates can be carried out by adding a trace amount of NAD.sup.+.
Dehydrogenation involving NAD.sup.+ as a coenzyme is generally a reversible reaction and the reaction is largely shifted toward the direction of NADH.fwdarw.NAD.sup.+ at neutral pH. In such a case, it is difficult to regenerate NAD.sup.+ in the dehydrogenase and NADH oxidase system and smoothly progress dehydrogenase reaction of the corresponding substrate. On the other hand, the dehydrogenase reaction is shifted toward the direction of NAD.sup.+ .fwdarw.NADH in an alkaline region, which regeneration of NAD.sup.+ is performed in coupling with NADH oxidase, whereby dehydrogeneration of the corresponding substrate readily proceeds.
As described above, in the case that dehydrogenase reaction is carried out in an industrial scale in the co-presence of dehydrogenase and NADH oxidase using NAD.sup.+ as coenzyme, it is important to develop NADH oxidase having a high activity even in an alkaline region and having a wide pH range of its action.
As NADH oxidases which oxidize NADH using molecular oxygen as an electron acceptor, there are hitherto reported oxidases derived from Lactobacillus plantarum (Agricultural and Biological Chemistry, 25, 876, 1961), derived from Streptococcus faecalis (Journal of Biological Chemistry, 237, 2647, 1962), derived from Acholeplasma laidlawii (European Journal of Biochemistry, 120, 329, 1981), derived from Bacillus megaterium (Journal of Biochemistry, 98, 1433, 1985), derived from Leuconostoc mesenteroides (Journal of Biochemistry, 97, 1279, 1985), etc.
However, NADH oxidases known in these techniques involve disadvantages that the microorganisms are cultured only with difficulty to make mass production difficult (Acholeplasma laidlawii, etc.); the enzyme is present in intracellular fraction so that it is difficult to harvest the enzyme and improve its content in the cells (Bacillus megaterium, etc.); the enzyme has poor heat stability so that it is difficult to expect that the enzyme could maintain its activity over a long period of time (Leuconostoc mesenteroides, etc.). Therefore, these enzymes are not suited or utilized for production in an industrial scale.